Project Summary The research objectives in this proposal are aimed at increasing our basic understanding of how Eya2 and Eya3, co-factors to the transcriptional activator Six1, may mimic their embryonic roles as they function in the muscle cancer rhabdomyosarcoma (RMS). With five year survival rates less than 30% and few targeted molecular therapies available, pediatric patients facing this deadly disease depend primarily on surgery and radiation for treatment. Long term effects of these treatments include severe, disabling and life-threatening effects (i.e. secondary tumors), which are experienced by more than 50% of childhood cancer survivors. Therefore, therapies with much lower toxicities, including those currently in development which are directed specifically at Eya-Six1, are of high interest, especially for young patients. However the functions of individual Eya family members in RMS progression, which will be investigated here, may affect whether these targeted therapies are appropriate for RMS patients. Previous studies of Eya proteins have demonstrated that biological context can have diverse effects on their function, as extreme as promoting tumor progression through enhancing cellular immortalization versus participating in tumor suppressive activities such as triggering apoptosis. Bioinformatic analysis of RMS gene expression indicates divergent roles for Eya proteins in this disease in particular, as Eya2 levels are significantly increased while Eya3 levels are significantly decreased in RMS tumors compared to normal muscle controls. This oppositional expression aligns with the dissimilar roles previously observed for these two Eya proteins in normal muscle development, in addition to recent evidence for varied immune roles. As embryonic programs are often usurped by tumor cells, this proposal is focused on investigating whether Eya2 and Eya3 function divergently in normal muscle and immune development and how these diverse roles may contribute to RMS progression by Eya2 versus RMS suppression by Eya3. Zebrafish will be utilized for these studies as CRISPR gene knockout strategies, ease of embryonal investigations and an existing RMS model all align to make this an ideal organism for these experiments. Data collected here has the potential to impact future drug design and expand treatment options for RMS patients.